Over 2% of the world's population is infected with hepatitis C virus (HCV), which causes severe liver diseases, including steatosis, progressive fibrosis, cirrhosis and hepatocellular carcinoma. Recently, a fully permissive HCV cell culture system using genotype 2a JFH1 has been developed. It is expected that this system will advance, by using a replicon system, the current HCV specific drug discovery effort. Subsequently, we developed an HCV infection system by using the clinically more relevant genotype 1a H77S. By taking advantage of this newly developed in vitro tissue culture model of HCV, we propose to elucidate the mechanism of NS2 in regulating the infectious virus production process. Our hypothesis that NS2 protein, which was known to contain a catalytic domain of NS2/NS3 protease, plays a critical role in HCV assembly and release is derived from our extensive preliminary studies using two different chimeras encoding H77 structural proteins within the background of JFH1. These chimeras showed distinct defects in the pathway leading to virus production. Our results suggest that the incompatibility of the NS2 sequence with its surrounding structural and nonstructural sequences is the major reason there is a defect in virus production, and the compensatory mutations accumulated at NS2 as well as other regions, including E1, p7 and NS3, could rescue infectious virus production. We also have more direct evidence that NS2 is involved in virus production and that NS2-specific viral assembly defect could be trans-complemented. In this proposal, we will first investigate if NS2 interacts with structural, as well as other nonstructural proteins, and, if that is the case, we will correlate the compensatory mutation-mediated phenotype of enhanced virus production with protein-protein interactions involving NS2. In our second aim, we will investigate the genotype-specific determinant(s) of assembly and release within the C-terminal region of NS2 by using recently available structural information within this region. In our third aim, we will characterize functional domain(s) of NS2 involved in viral assembly and maturation by analyzing specific properties of NS2 related to virus production and by delineation of the domain(s) involved in these processes using a trans-complementation assay. The data generated from this proposal will advance our knowledge of the mechanism of infectious virus production. Moreover, if the specific role of NS2 involved in virus production is elucidated through this proposal, it will make possible the development of specific inhibitors against the dual functions of HCV NS2 involved in both viral RNA replication and viral particle formation. PUBLIC HEALTH RELEVANCE: Hepatitis C Virus (HCV) infection leads to public health problems by causing severe liver diseases. Our preliminary data indicate that HCV NS2 protein, known to contain a catalytic domain of NS2/NS3 protease, plays a critical role in HCV assembly and release. The data generated from our proposal will advance our knowledge related to HCV virus production and make possible the development of specific inhibitors against the dual functions of HCV NS2 involved in both viral RNA replication and viral particle formation.